The long term goal of this work is to understand the molecular basis for the regulation of Arp2/3 complex. Arp2/3 complex is an essential component of the actin assembly machinery because of its ability to nucleate branched actin filaments in response to cellular signals. Arp2/3 complex is involved in a number of processes that occur in healthy cells, such as endocytosis and motility of growth cones, but also plays roles in host cell infection by bacterial pathogens and the metastasis of tumor cells. The activity of Arp2/3 complex is tightly regulated in vivo, and numerous activators and inhibitors of the complex have been discovered, including WASp/Scar family proteins and coronin family proteins. Despite the central importance of the Arp2/3 complex, the molecular mechanism of its activation is still unknown due to the lack of biochemical and high resolution structural information of partially or fully activated states of the complex. Here we propose to use structural, biochemical and cell biological approaches to determine a precise molecular mechanism by which Arp2/3 complex and its regulatory proteins create branched actin networks. We will pursue this goal through the following three specific aims: 1.) Determine the key structural elements that allow Arp2/3 complex to bind to mother and daughter filaments of actin; 2.) Determine how WASp/Scar family proteins activate Arp2/3 complex; and 3.) Determine how Coronin proteins inhibit Arp2/3 complex. Our approach will be to use mutational analysis and biochemical assays to precisely map the interactions of S. pombe Arp2/3 complex with mother and daughter filaments of actin. We will use mass spectrometry, x-ray crystallography and mutational analysis to determine where WASp/Scar family proteins bind to Arp2/3 complex and how they recruit the first actin monomer for the daughter filament. Finally, we will use biochemical and structural methods to determine how yeast coronin proteins inhibit Arp2/3 complex and influence its interactions with actin filaments.